Germline and somatic <i>RUNX1</i> variants in a pediatric bone marrow failure cohort
Authors: Ayako Yamamori, Motoharu Hamada, Hideki Muramatsu, Manabu Wakamatsu, Asahito Hama, Atsushi Narita, Yusuke Tsumura, Taro Yoshida, Takehiko Doi, Kazuki Terada, Takeshi Higa, Nobuyuki Yamamoto, Hiroki Miura, Mitsutaka Shiota, Kenichiro Watanabe, Nao Yoshida, Ryo Maemura, Masayuki Imaya, Shunsuke Miwata, Kotaro Narita, Shinsuke Kataoka, Rieko Taniguchi, Kyogo Suzuki, Nozomu Kawashima, Nobuhiro Nishio, Hideto Iwafuchi, Masafumi Ito, Seiji Kojima, Yusuke Okuno, Yoshiyuki Takahashi
Journal: American Journal of Hematology (2023)

To the Editor: RUNX1 is a key regulatory gene involved in hematopoiesis and myeloid differentiation.1 Its somatic variants play essential roles in the pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).1, 2 Furthermore, its heterozygous germline variants (loss-of-function, leading to haploinsufficiency or dominant-negative variants) are proven to be a major cause of familial platelet disorder with associated myeloid malignancy (FPD-MM), an autosomal dominant genetic disorder characterized by a mild to moderate thrombocytopenia and an abnormal platelet function.1, 2 In addition, approximately 40% of patients with FPD-MM develop hematological malignancy during their lifetime.2 FPD-MM, together with ANKRD26 and ETV6 germline variants, was listed as “myeloid neoplasms with germline predisposition and pre-existing platelet disorder” in the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors.3 Diagnosis of FPD-MM is difficult without genetic analysis, especially in sporadic pediatric patients generally due to the absence of specific physical symptoms or abnormalities. To clarify the clinical significance of RUNX1 variants in pediatric patients with bone marrow failure (BMF), we retrospectively analyzed the clinical information of nine patients (2.8%) with pathogenic RUNX1 variants (germline [n = 7] and somatic [n = 2]) out of 324 patients in the pediatric BMF registry. A total of 324 patients with clinically diagnosed or suspected BMF were enrolled in our pediatric BMF registry between December 2013 and May 2019 (Supplemental Table 1). We extracted the genomic DNA from the peripheral blood (PB), oral mucosa, and nails and conducted targeted capture sequencing and/or Sanger sequencing. All pathogenic variants of BMF-associated genes, including RUNX1, were determined as described in Supplemental Methods. The clinical diagnoses of the patients are presented in Supplemental Table 2. When available, morphological evaluation of PB and bone marrow (BM) smear specimens from patients with RUNX1 variants was performed by physicians with expertise in pediatric hematological morphology (AH, MI, and HI). Detailed definitions of morphological evaluation are described in Supplemental Methods. Subsequently, we conducted a systematic literature review of publications describing patients with FPD-MM and their information on BM morphology before leukemic transformation (Supplemental Figure 1). Detailed procedures are described in the Supplemental Methods. In this study, 9 patients (2.8%) among the 324 pediatric BMF cohort had RUNX1 variants (Supplemental Table 1). We described their detailed medical histories in Supplemental Notes and genetic trees in Supplemental Figure 2. Seven patients (four boys and three girls) had germline RUNX1 variants, whereas two girls had somatic RUNX1 variants. Eight patients and one patient (UPN-1021b) had thrombocytopenia before and after diagnosis, respectively, with a median platelet count at diagnosis of 36.0 × 109/L (range: 5.0–92.0 × 109/L). Their clinical diagnoses before the genetic tests were classified as follows: thrombocytopenia (n = 5), inherited BMF syndrome, not otherwise specified (n = 3), and asymptomatic (n = 1). Upon diagnosis, the median age was 8 months (range: 0–10 years), and four of the patients were enrolled shortly after birth. Five patients from four pedigrees with a family history of thrombocytopenia were identified with a unique patient number (UPN) as follows: UPN-289, UPN-1013, UPN-1021a, UPN-1021b, and UPN-1507. Of note, the grandfather of UPN-1013 had thrombocytopenia followed by MDS. UPN-934 and UPN-1128 had a medical history of laryngomalacia. Furthermore, no other physical abnormalities were observed among the patients, except for UPN-934 who had short stature. Figure 1A and Supplemental Table 3 summarize the genetic diagnoses of all nine patients who presented with heterozygous pathogenic variants: seven with germline variants (two deletions [exons 4–7], two frameshift variants [p.Pro357ArgfsTer129 and p.Leu381ArgfsTer105], one nonsense variant [p.Glu422*], one splice site variant [c.351 + 1G > A], one missense variant [p.Thr196Ala]), and two nonsense somatic variants [p.Arg204* and p.Arg201*]. The functional consequences of RUNX1 variants were classified as dominant negative (DN) in UPN-1596, UPN-934, and UPN-1334, and loss-of-function (LOF) in UPN-1013, UPN-1128, UPN-1507, UPN-1221a, UPN-1221b, and UPN-289, respectively (Supplemental Methods). Three of the nine patients developed hematological malignancies: T-LBL (UPN-1507) and MDS (UPN-934 and UPN-1334). The first had a germline RUNX1 variant, whereas the others had somatic RUNX1 variants. Hematological morphology was evaluated in seven patients with available PB (n = 6) or BM (n = 5) smear (Figure 1B–F, Supplemental Table 4). Regarding the PB-cell morphology, five patients showed normal platelets and only one patient (UPN-1596 with germline RUNX1 variant) had large platelets. Regarding BM cellularity, four patients had normocellular marrow (germline [n = 3] and somatic [n = 1]) and one patient had hypocellular marrow (UPN-1021a with germline variant). Regarding megakaryocyte counts, two patients (both with germline) had normal megakaryocytes and three patients had decreased megakaryocytes (germline [n = 2] and somatic [n = 1]). Megakaryocyte dysplasia was observed in all four evaluable patients with germline RUNX1 variant and in a patient with somatic RUNX1 variant (UPN-1334). Trilineage dysplasia was observed in two patients with DN-type RUNX1 variants (UPN-1596 and UPN-1334). We summarized clinical information with PB and BM morphology evaluation in 13 patients with hematological disorders with germline RUNX1 variants identified in a systematic literature review (Supplemental Tables 5, 6). The median age at diagnosis was 34 years (range: 0–70 years), with five children and eight adults. Megakaryocyte dysplasia was a common finding in all patients. In addition to megakaryocyte dysplasia, H-II-3 with DN-type RUNX1 variant was reported to harbor dysplasia in erythroid cells and G-I-1 with LOF-type RUNX1 variant in myeloid cells, respectively. Remarkably, one patient was malignantly transformed into MDS (H-II-3) and another into AML (M-II-1) (Figure 1G). In a consecutive cohort study of 324 children with BMF, we identified pathogenic RUNX1 variants in nine patients, of whom seven (78%) were diagnosed with FPD-MM and two (22%) with RUNX1 somatic variants with exclusive initial symptoms attributed to pediatric thrombocytopenia. If based solely on clinical symptoms and family history, it would be difficult to determine whether the identified RUNX1 variants in children with BMF as well as in adults with MDS/AML are of germline or somatic origins. Therefore, genetic analysis of nonhematological tissue-derived DNA is required. In our pediatric cohort study, one of seven children with FPD-MM developed T-LBL at the age of 7 years. Although FPD-MM patients usually develop hematological malignancies during adulthood, attention should be paid to any possible malignant transformation during childhood. In our study, three of seven children with FPD-MM exhibited thrombocytopenia in the neonatal period. In line with these results, Bagla et al. also reported an infantile patient with FPD-MM with thrombocytopenia.4 Conversely, Homan et al. reported 167 patients with FPD-MM enrolled in the interactive public international collaborative database for RUNX1 (RUNX1db) but did not include patients younger than 1 year.1 Although neonatal thrombocytopenia is mainly caused by an increased platelet destruction due to immune processes and platelet activation and consumption,5 hereditary thrombocytopenia, including FPD-MM, is extremely rare and is not generally considered as a differential diagnosis of neonatal thrombocytopenia. Hence, consideration of the RUNX1 gene mutation analysis in neonatal thrombocytopenia is likely to improve the chances of FPD-MM diagnosis. Only a few studies have evaluated detailed blood cell morphologic abnormalities in patients with FPD-MM; however, normal-sized platelets and small megakaryocytes have been reported as characteristic findings in our study (Figure 1G). Fournier et al. evaluated comparative morphological features in patients with hereditary thrombocytopenia due to RUNX1, ANKRD26, and ETV6 genes. In this context, they reported that only FPD-MM patients with germline RUNX1 variants had small megakaryocytes with separated nuclei.6 Consistent with these reports, our study demonstrated that the common hematologic morphologic finding in FPD-MM had normal-sized platelets and megakaryocyte dysplasia (Figure 1C–G). Additionally, trilineage dysplasia was observed in one patient with a DN-type RUNX1 variant (UPN-1596), without dysplasia other than the megakaryocytic lineage observed in six patients with LOF-type RUNX1 variants. However, a larger prospective study with central morphological diagnosis is needed to evaluate a genotype–phenotype correlation between RUNX1 variant classification and morphological findings. On another side, although micromegakaryocyte is a characteristic finding of megakaryocyte dysplasia commonly observed in MDS, only one patient with FPD-MM was reported as having micromegakaryocyte in previous studies (Figure 1G, Supplemental Table 6). In contrast, micromegakaryocytes were present in three of the four FPD-MM patients for whom we were able to evaluate BM images (Figure 1G). Further studies are necessary to determine whether the presence of micromegakaryocytes is a universal BM finding in FPD-MM patients who have not clinically progressed to MDS/AML. This study has several limitations. First is the long-term prognosis, including malignant transformation rate, which was not adequately assessed because the cohort analysis was mainly limited to the childhood period of observation. Second, only two pediatric patients had somatic RUNX1 variants; thus, their hematological morphology is not fully comparable to that of FPD-MM patients. In conclusion, genetic analyses of germline and somatic RUNX1 variants and the evaluation of their hematological morphology have a significant clinical value in the differential diagnosis of pediatric patients with BMF. In particular, genetic analysis should be conducted in neonates with thrombocytopenia because inherited blood disorders, including FPD-MM, may be underdiagnosed in neonatal patients. The authors acknowledge all clinicians, patients, and their families, and thank Ms. Yoshie Miura, Ms. Fumiyo Ando, Ms. Hiroko Ohno, and Ms. Chie Amahori for their valuable assistance. This study was supported by the Nagoya Pediatric Cancer Fund. The authors report no potential conflict of interest. Written informed consent was obtained from all parents or legal guardians before the genetic tests were conducted. Data S1. Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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